Cross effects of heat stress and three insecticides on the survival of the codling moth Cydia pomonella (L.): Investigating the molecular and biochemical mechanisms.

As temperature is expected to strongly increase in the future, understanding temperature-mediated toxicity of insecticides is determinant to assess pest management efficiency in a warming world. Investigating molecular and biochemical mechanisms associated with cross mechanisms of temperature and insecticides on pests' tolerance would also be useful in this context. This study aimed to investigate cross effects between temperature and insecticides on the survival of a major pest, the codling moth Cydia pomonella, and their underlying mechanisms. The effect of three insecticidal active ingredients, i.e. chlorantraniliprole, emamectin and spinosad, was assessed at different temperatures on: (i) C. pomonella larval survival; (ii) detoxification enzymes activities (cytochrome P450 multi-function oxygenases, carboxylesterases and glutathione S-transferases) and (iii) genes expression of some detoxification enzymes, heat shock proteins and receptors targeted by the insecticides. We observed a decreased efficiency of emamectin and spinosad at high temperature to control the codling moth while no influence of temperature on chlorantraniliprole efficacy was observed. Detoxification enzymes activities were improved by heat stress alone but not by double stress (temperature + insecticides). Moreover, two detoxification genes (Cyp9A61 and Gst1) were over-expressed by a single stress but not by two stresses while Hsp70 and Cyp6B2 genes may be involved in tolerance to two stresses in C. pomonella. These results confirmed the cross effects of temperature and insecticides on C. pomonella for emamectin and spinosad and provided clues to understand how temperature affects the susceptibility of C. pomonella to insecticides. They illustrate however the complexity of molecular and biochemical responses of individuals facing multiple stresses.

Regulation of sugar metabolism genes in the nitrogen-dependent susceptibility of tomato stems to Botrytis cinerea.

BACKGROUND AND AIMS: The main soluble sugars are important components of plant defence against pathogens, but the underlying mechanisms are unclear. Upon infection by Botrytis cinerea, the activation of several sugar transporters, from both plant and fungus, illustrates the struggle for carbon resources. In sink tissues, the metabolic use of the sugars mobilized in the synthesis of defence compounds or antifungal barriers is not fully understood. METHODS: In this study, the nitrogen-dependent variation of tomato stem susceptibility to B. cinerea was used to examine, before and throughout the course of infection, the transcriptional activity of enzymes involved in sugar metabolism. Under different nitrate nutrition regimes, the expression of genes that encode the enzymes of sugar metabolism (invertases, sucrose synthases, hexokinases, fructokinases and phosphofructokinases) was determined and sugar contents were measured before inoculation and in asymptomatic tissues surrounding the lesions after inoculation. KEY RESULTS: At high nitrogen availability, decreased susceptibility was associated with the overexpression of several genes 2 d after inoculation: sucrose synthases Sl-SUS1 and Sl-SUS3, cell wall invertases Sl-LIN5 to Sl-LIN9 and some fructokinase and phosphofructokinase genes. By contrast, increased susceptibility corresponded to the early repression of several genes that encode cell wall invertase and sucrose synthase. The course of sugar contents was coherent with gene expression. CONCLUSIONS: The activation of specific genes that encode sucrose synthase is required for enhanced defence. Since the overexpression of fructokinase is also associated with reduced susceptibility, it can be hypothesized that supplementary sucrose cleavage by sucrose synthases is dedicated to the production of cell wall components from UDP-glucose, or to the additional implication of fructose in the synthesis of antimicrobial compounds, or both.

Molecular Tools for the Detection and the Identification of Hymenoptera Parasitoids in Tortricid Fruit Pests.

Biological control requires specific tools for the accurate detection and identification of natural enemies in order to estimate variations in their abundance and their impact according to changes in environmental conditions or agricultural practices. Here, we developed two molecular methods of detection based on PCR-RFLP with universal primers and on PCR with specific primers to identify commonly occurring larval parasitoids of the tortricid fruit pests and to estimate parasitism in the codling moth. Both methods were designed based on DNA sequences of the COI mitochondrial gene for a range of parasitoids that emerged from Cydia pomonella and Grapholitamolesta caterpillars (102 parasitoids; nine species) and a range of potential tortricid hosts (40 moths; five species) damaging fruits. The PCR-RFLP method (digestion by AluI of a 482 bp COI fragment) was very powerful to identify parasitoid adults and their hosts, but failed to detect parasitoid larvae within eggs or within young C. pomonella caterpillars. The PCR method based on specific primers amplified COI fragments of different lengths (131 to 463 bp) for Ascogaster quadridentata (Braconidae); Pristomerusvulnerator (Ichneumonidae); Trichomma enecator (Ichneumonidae); and Perilampus tristis (Perilampidae), and demonstrated a higher level of sensibility than the PCR-RFLP method. Molecular estimations of parasitism levels in a natural C. pomonella population with the specific primers did not differ from traditional estimations based on caterpillar rearing (about 60% parasitism in a non-treated apple orchard). These PCR-based techniques provide information about within-host parasitoid assemblage in the codling moth and preliminary results on the larval parasitism of major tortricid fruit pests.

How varying parameters impact insecticide resistance bioassay: An example on the worldwide invasive pest Drosophila suzukii.

Monitoring pesticide resistance is essential for effective and sustainable agricultural practices. Bioassays are the basis for pesticide-resistance testing, but devising a reliable and reproducible method can be challenging because these tests are carried out on living organisms. Here, we investigated five critical parameters and how they affected the evaluation of resistance to the organophosphate phosmet or the pyrethroid lambda-cyhalothrin using a tarsal-contact protocol on Drosophila suzukii, a worldwide invasive pest. Three of the parameters were related to insect biology: (i) sex, (ii) age of the imago (adult stage) and (iii) genetic diversity of the tested population. The two remaining parameters were linked to the experimental setup: (iv) the number of individuals tested per dose and (v) the duration of exposure to the active ingredient. Results showed that response to insecticide differed depending on sex, males being twice as susceptible to phosmet as females. Age principally affected young females' susceptibility to phosmet, because 0-24 hour-old flies were twice as susceptible as 24-48 hour-old and 72-96 hour-old females. Genetic diversity had no observable effect on resistance levels. The precision and accuracy of the median lethal dose (LD50) were greatly affected by the number of individuals tested per dose with a threshold effect. Finally, optimal duration of exposure to the active ingredient was 24 h, as we found an underestimation of mortality when assessed between 1 and 5 h after exposure to lambda-cyhalothrin. None of the main known point mutations on the para sodium channel gene associated with a knockdown effect were observed. Our study demonstrates the importance of calibrating the various parameters of a bioassay to develop a reliable method. It also provides a valuable and transferable protocol for monitoring D. suzukii resistance worldwide.

Limited genetic structure and demographic expansion of the Brassicogethes aeneus populations in France and in Europe.

BACKGROUND: The pollen beetle, Brassicogethes aeneus (Fabricius, 1775), is one of the most significant pests of oilseed rape. To shed light on past and current pollen beetle demography (dispersal, population size), 12 microsatellite markers were developed, and population genetic diversity and structure were analysed at different spatial scales in France and in Europe from 433 individuals collected in 18 winter oilseed rape fields. RESULTS: Genetic differentiation among the population samples was low but was significant between the Estonian sample and the rest of Europe. Isolation by distance was significant only at the European scale. Genetic variability was similar among the 18 population samples. Demographic inferences suggested a recent expansion of B. aeneus population size over Europe, possibly corresponding to an increase in oilseed rape crop area during past decades. CONCLUSION: Current population size and dispersal are not straightforward to estimate from the distribution of genetic variability in B. aeneus over Europe because of the complexity of the demographic history of this pest. Nevertheless, because gene flow was important enough to prevent strong genetic differentiation at large geographical scales, the management of pollen beetle populations should likely be thought of at a continental Europe level. © 2018 Society of Chemical Industry.

A chromosome-level genome assembly of Cydia pomonella provides insights into chemical ecology and insecticide resistance.

The codling moth Cydia pomonella, a major invasive pest of pome fruit, has spread around the globe in the last half century. We generated a chromosome-level scaffold assembly including the Z chromosome and a portion of the W chromosome. This assembly reveals the duplication of an olfactory receptor gene (OR3), which we demonstrate enhances the ability of C. pomonella to exploit kairomones and pheromones in locating both host plants and mates. Genome-wide association studies contrasting insecticide-resistant and susceptible strains identify hundreds of single nucleotide polymorphisms (SNPs) potentially associated with insecticide resistance, including three SNPs found in the promoter of CYP6B2. RNAi knockdown of CYP6B2 increases C. pomonella sensitivity to two insecticides, deltamethrin and azinphos methyl. The high-quality genome assembly of C. pomonella informs the genetic basis of its invasiveness, suggesting the codling moth has distinctive capabilities and adaptive potential that may explain its worldwide expansion.

Diversity of insecticide resistance mechanisms and spectrum in European populations of the codling moth, Cydia pomonella.

Only a few of the registered insecticides against Cydia pomonella L. are still effective in areas where insecticide resistance has emerged in this pest. Resistance mechanisms are multiple, and their lone or cumulative effects in a single population are not completely understood. A detailed estimation of resistance spectrum is still required to define the suitable insecticides to use against a given population. The efficacy of ten insecticides was therefore investigated together with the resistance mechanisms expressed in four laboratory strains and 47 field populations of C. pomonella from five countries. Bioassays were performed using topical applications of diagnostic concentrations on diapausing larvae, and resistance mechanisms were analysed on adults emerging from control insects. All populations exhibited a reduced susceptibility to at least one insecticide when compared with the susceptible laboratory strain. Cross-resistances were observed between azinphos-methyl or phosalone and more recent compounds such as spinosad and thiacloprid. Resistances to azinphos-methyl, diflubenzuron, spinosad, tebufenozide and thiacloprid were significantly correlated with mixed-function oxidase activity, while increased glutathione-S-transferase and reduced non-specific esterase activities were correlated with resistance to azinphos-methyl and emamectin, respectively. Conversely, resistances to azinphos-methyl, tebufenozide and thiacloprid were negatively correlated with increased esterase activity. None of the observed mechanisms explained the loss of susceptibility of populations to chlorpyrifos-ethyl, and no significant correlation was detected between resistance to deltamethrin and the presence of the kdr mutation. The suitability of such non-target instars to monitor insecticide resistance in field populations is discussed.

Biochemical and Molecular Mechanisms Associated With the Resistance of the European Corn Borer (Lepidoptera: Crambidae) to Lambda-Cyhalothrin and First Monitoring Tool.

The European corn borer (Ostrinia nubilalis (Hübner)) is one of the most serious corn pest in Europe where it is controlled with pesticides, in particular, pyrethroids. First control failures with this chemical family occurred on the field in 2008 in the center of France, and the first resistance case was described in 2012. In the present study, we investigate resistance mechanisms involved in seven French populations of O. nubilalis collected in the field. Resistances to deltamethrin and lambda-cyhalothrin were confirmed, with a higher resistance ratio for lambda-cyhalothrin (63.79 compared to 7.67). Resistance to the two active compounds was correlated except for one population, indicating a high probability of cross-resistance. Analyses of the activity of three major families of detoxification enzymes in resistant individuals showed a significant increase of the average MFO activity in males of four populations (activity ratios of 2.76-5.73) and higher GST activity in females of two other populations (activity ratios 4.48 and 5.21). Molecular investigation of the sodium channel gene sequence showed the presence of the kdr mutation in a highly resistant individual. We designed a PCR-RFLP screening tool to search for this mutation in the field, and we found it in five populations but not in the susceptible one. The resistance of O. nubilalis to pyrethroids in France seems to result from a combination of resistance mechanisms, possibly as a consequence of a selection pressure with an exceptional duration (almost 40 yr old).

Multiple origins of the sodium channel kdr mutations in codling moth populations.

Resistance to insecticides is one interesting example of a rapid current evolutionary change. DNA variability in the voltage-gated sodium channel gene (trans-membrane segments 5 and 6 in domain II) was investigated in order to estimate resistance evolution to pyrethroid in codling moth populations at the World level. DNA variation among 38 sequences revealed a unique kdr mutation (L1014F) involved in pyrethroid resistance in this gene region, which likely resulted from several convergent substitutions. The analysis of codling moth samples from 52 apple orchards in 19 countries using a simple PCR-RFLP confirmed that this kdr mutation is almost worldwide distributed. The proportions of kdr mutation were negatively correlated with the annual temperatures in the sampled regions. Homozygous kdr genotypes in the French apple orchards showed lower P450 cytochrome oxidase activities than other genotypes. The most plausible interpretation of the geographic distribution of kdr in codling moth populations is that it has both multiple independent origins and a spreading limited by low temperature and negative interaction with the presence of alternative resistance mechanisms to pyrethroid in the populations.